**4. Importance of infectious diseases in Uruguayan pinniped colonies with zoonotic potential**

It has been documented that the introduction of novel pathogens into a native animal population without previous exposure could result in epizootics. Human and wildlife populations share a wide range of diseases. While the most common disease transmissions reported are between wild and domestic animals (though many zoonoses do have wildlife origin), emerging diseases of animal origin represent one of the greatest potential threats to public health.

The emergence and re-emergence of over 30 agents have been reported in marine mammals (Miller et al., 2001). There are several reports about their susceptibility to virus, bacterial, fungal and parasitic agents, provoking diseases that result in mass mortality events (Gulland et al., 1996; Miller et al., 2004; Ostehaus, 2000). Once new or previous pathogens are established in the host, they can represent a health risk to other marine vertebrates, humans, or both. It is, however, very difficult to know which routes these zoonotic marine mammal infections take in the marine environment.

168 New Approaches to the Study of Marine Mammals

animals.

public health.

**with zoonotic potential** 

animals, but in wild conditions these procedures are impractical. Chest radiographs were performed on pinnipeds of different sizes, but in cases of very small lesions in large animals with thick blubber, radiological images do not give appropriate information (Forshaw & Phelps, 1991; Jurczynski et al., 2012). In some zoos computer tomography has been used for detection of small calcified granulomas (Jurczynski et al., 2011, 2012), but it is very difficult to be used routinely. Different serological tests (rapid test, Elephant TB STAT-PAK, Chembio; multiantigen print immunoassay (MAPIA) Chembio; dual path platform assay (DPP Vet; Chembio) have been used in *O. flavescens* individuals in captivity, with DPP technique demonstrating greater sensitivity (87.5%) (Jurczynski et al., 2012). The tuberculin skin test (TST) with purified protein derivative (PPD bovine and avian) for screening has been done in *O. flavescens* and *A. australis* individuals (Bernardelli et al., 1990; Castro-Ramos et al., 1998; Kiers et al, 2008; Lacave, 2009) and reported by Forshaw & Phelps (1991) in *A. forsteri, A. pusillus doriferus* and *N. cinerea*. This technique is very sensitive, economical and easy to perform. The possible occurrence of false negatives must be taken into consideration in cases of advanced infection with anergy or very recent infections that have not yet generated an appropriate immune response (Jurczynski et al., 2011). False positives may also occur in nonspecific cases of exposure to non-tuberculous mycobacteria (*Mycobacterium avium, M. chelonae, M. fortuitum* and *M. smegmatis*) (Bernardelli et al., 1990; Forshaw & Phelps, 1991). Tissue and bronchial secretion smears have been used for diagnosis (Jurczynski et al., 2012), but the AFB may correspond to mycobacteria other than *M. pinnipedii* and, therefore, other confirmation methods must be used. Molecular techniques (PCR, spoligotyping and MIRU / VNTR) applied to samples from purified cultures, tissues and sputum, have produced quick results, allowing the identification of strains involved and their origin (Cousins et al., 2003; Jurczynski et al., 2011; Kiers et al., 2008; Kriz et al., 2011; Moser et al., 2008). In zoo collections, it is suggested that the final diagnosis should be based on the simultaneous use of three different methods, needing a minimum of two positive tests to increase the overall sensitivity when making the final decision for euthanasia. In case of wild animals, the possible diagnostic methods could include the TST, culture and molecular analysis of sputum (PCR) and serology (DPP). This would be extremely important in order to establish or confirm the endemic conditions of the disease in wild colonies and prevent the exportation or handling of carrier

**4. Importance of infectious diseases in Uruguayan pinniped colonies** 

It has been documented that the introduction of novel pathogens into a native animal population without previous exposure could result in epizootics. Human and wildlife populations share a wide range of diseases. While the most common disease transmissions reported are between wild and domestic animals (though many zoonoses do have wildlife origin), emerging diseases of animal origin represent one of the greatest potential threats to

The emergence and re-emergence of over 30 agents have been reported in marine mammals (Miller et al., 2001). There are several reports about their susceptibility to virus, bacterial, The dynamic governing the relationship between infectious diseases affecting humans and marine vertebrates, including sea mammals is very complex and generally poorly understood. It is probable that human activity is a greater threat to marine vertebrate health than vice versa (Mos et al., 2006). However, it is very difficult to establish the role of marine animals as vectors or carriers of zoonotic diseases. Although the role of sea mammals in transmission of potential zoonotic pathogens is not well established, several risk factors, including frequent and prolonged direct contact with live specimens, were clearly identified in workers exposed to these animals (Hunt et al. 2008). Thus, these animals may present a zoonotic potential and also the potential for epizootic events which could cause health problems in marine animals. Since the influenza virus is transmitted by direct contact with infected individuals, by aerosol, or contact with infected objects, several incidents of influenza A virus transmission have occurred from infected seals and whales maintained in captivity to humans (Webster et al.*,* 1981*).*

Due to their life span, high trophic feeding and the continuous exposition to emerging pathogens, sea mammals might be considered as sentinel species of emerging/re-emerging diseases. Pinnipeds share these characteristics and may serve as effective sentinels providing information about public and aquatic ecosystem health, and indicating the current or potential negative impacts on animal health at the individual or population level.

Implementing a proper management and the accurate execution of conservation policies of wildlife requires the analysis of the vulnerability of the animals to infectious diseases*.* The majority of animals included in our studies belonged to colonies near cities or towns in proximity to shore (Punta del Este, Polonio's Cape), regions that are commonly used by humans, marine and terrestrial mammals and different avian species, establishing an optimal opportunity for zoonotic disease transmission and long-term disease maintenance. The dense aggregations of pinniped colonies make fur seals and sea lions vulnerable to epizootics. Also, movements between adjacent latitudinal domains are common in both pinniped species, which could transmit or acquire pathogens during feeding trips to other parts of the Atlantic Ocean and the La Plata River.

The diagnosis of influenza virus in high-density seal populations in Uruguay generated considerable concern about the potential impacts on South American fur seal and sea lion colonies, as well as potential health risks to humans and domestic animals. In the evolution and ecology of influenza viruses, interspecies transmission is an important factor; seabirds and marine mammals are conspicuous animals that integrate changes in the ecosystem and reflect the existing state of the environment (Aguirre & Tabor, 2004; Boersma, 2008; Moore, 2008; Thiele et al., 2004). Transmission of the influenza virus occurs between avian and several marine mammal species *(*Mandler et al.*,* 1990*)* at least for influenza A virus,

representing an important step in the evolution and emergence of new mammalian viral strains. Fur seals have the potential to serve as an influenza reservoir for other mammal species. However, more detailed studies are needed to elucidate the role of seals in the epidemiology of influenza along the Uruguayan coasts and in other South American countries.

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 171

quickly due to their weight and reach the ground without evaporation. Smaller drops evaporate and decrease in size, becoming infectious droplet nuclei, which remain suspended in ambient air for a long time and can be airborne for days. Successful transmission requires that these micro droplets charged with bacilli are of sufficiently small size (1 to 5 µm) to enable them to reach very deep into the lungs and alveoli (Rider, 1999). This phenomenon is more frequent in captive conditions in zoos and aquaria where spray released during enclosure hygiene, poor ventilation and exposure among animals in confined environments, constitute increased risk factors for infection as has been shown in different zoos (Cousins et al., 1993 and 2003; Kiers et al., 2008; Thompson et al., 1993). In wild pinniped colonies, natural ventilation and increased exposure to UV is a natural way of microorganism control that could be the reason for the less common occurrence of TB in

In the early course of host-pathogen interaction, mycobacteria are phagocytized by alveolar macrophages. In case the macrophage cannot destroy the pathogen, it resides in a quiescent state with a relatively low multiplication until cell mediated immunity is compromised transiently or permanently. Among the risk factors for immune-compromise, are age, history of a spontaneous TB cure with residual fibrotic lesions, and the time elapsed since infection. Other medical conditions such as endocrine disorders, tumors, malnutrition and stress may influence the progression of infection to disease (Musser et al., 2000; Rider, 1999). This evolution of the disease is consistent with the findings by different authors who have isolated AFB in organs without lesions from pups and juveniles (Bruning-Fann et al., 2001), identified the presence of calcified granulomas in mediastinal lymph nodes in adult and some juvenile animals in captivity and documented tuberculosis lesions in older animals

Keepers and veterinarians at zoos, aquaria and rehabilitation centers are at increased risk of infection because of their extensive contact with the animals. The degree of risk depends on the type of accommodation and sanitizing procedures of the enclosures (Kiers et al., 2008; Thompson et al., 1993) in association with the presence of open tuberculosis cases. Given the zoonotic condition of the disease, it is important to take preventive measures in all personnel working near or with access to wild and captive pinnipeds (researchers, fishermen, seal hunters, rehabilitation centers, aquaria and zoos staff) (Kiers et al., 2008; Thompson et al., 1993) and to establish hygiene measures for reducing the chances of

Since development of the disease is triggered under immunosuppressive conditions, it is important to assess which factors directly or indirectly affect the immune system and allow development of the disease in wild populations of the Southern Hemisphere. This also applies to other diseases that may be affecting the health status of wild colonies, particularly

Further investigation is needed in order to establish the sources of zoonotic potential in the marine environment and to better understand the nature of health risks for sea mammals and human. Additional epidemiologic studies are also required, to investigate epizootic

in *O. flavescens* from Uruguay, whose population is declining.

these colonies.

(Jurczynski et al., 2011, 2012).

spreading the infection.

Our findings confirm that fur seals can act as reservoirs of human influenza strains that circulated in the past, and also suggest that influenza A and B viruses may be transmitted from humans to seals as has been mentioned by other authors (Ohishi et al., 2002, 2004; Osterhaus, 2000). This transmission is due to the highly social lifestyle of pinnipeds, which congregate at sea and on land, and frequently associate with seals from other colonies. It is important to note that there is a strong interaction between seals and humans on Lobos Island and Polonio's Cape during live animal captures; nowadays, most interactions occur during capture and research activities, as well as in rehabilitation centers and sometimes with divers that swim near the seal islands. These events constitute opportunities for new influenza strains to jump between humans and seals, providing the potential for an epidemic event. Gaining information on the full spectrum of influenza viruses circulating in our seal colonies and detecting these viruses will remain an important task for its surveillance, outbreak control, and animal and public health.

As described previously, tuberculosis in pinnipeds had been recorded from the beginning of the twentieth century but it was not until mid 80's and later that several publications appeared with data from stranded animals and zoological collections. It is important to note that most records are from Otariids from the Southern Hemisphere, a single diagnosis is from a southern elephant seal and one from one native Northern Otariid species kept in captivity in the Northern Hemisphere.

Despite the fact that the seal harvest in Uruguay extended for 200 years, there are no records of macroscopic lesions observed in animals, nor of tuberculosis diagnosis from the staff working in the capture, slaughter or processing of the by-products, that could contribute retrospective data to this disease in Uruguayan pinniped colonies. The disease is considered endemic because of the numerous cases diagnosed in wild animals and the high prevalence of South American seals kept in aquaria and zoos. Unfortunately, to date non epidemiological studies have been conducted in any of the seal colonies from the Southern Hemisphere which could indicate the prevalence of the disease in its natural environment.

Information presented in this work, including the isolation of *M. pinnipedii* in pups and juvenile seals, indicates the early transmission of the organism. However, it is difficult to establish the course of the disease or immune mechanisms that may develop in each individual to control the infection or, in other cases, allow individuals to act as healthy carriers for several years until some trigger factor determines the development of the disease.

The most important mechanism of transmission of *M. tuberculosis*, and probably *M. pinnipedii*, is through the air by droplets produced when an individual with respiratory tract TB eliminates aerosolized microorganisms by coughing or sneezing. Large droplets fall quickly due to their weight and reach the ground without evaporation. Smaller drops evaporate and decrease in size, becoming infectious droplet nuclei, which remain suspended in ambient air for a long time and can be airborne for days. Successful transmission requires that these micro droplets charged with bacilli are of sufficiently small size (1 to 5 µm) to enable them to reach very deep into the lungs and alveoli (Rider, 1999). This phenomenon is more frequent in captive conditions in zoos and aquaria where spray released during enclosure hygiene, poor ventilation and exposure among animals in confined environments, constitute increased risk factors for infection as has been shown in different zoos (Cousins et al., 1993 and 2003; Kiers et al., 2008; Thompson et al., 1993). In wild pinniped colonies, natural ventilation and increased exposure to UV is a natural way of microorganism control that could be the reason for the less common occurrence of TB in these colonies.

170 New Approaches to the Study of Marine Mammals

countries.

disease.

representing an important step in the evolution and emergence of new mammalian viral strains. Fur seals have the potential to serve as an influenza reservoir for other mammal species. However, more detailed studies are needed to elucidate the role of seals in the epidemiology of influenza along the Uruguayan coasts and in other South American

Our findings confirm that fur seals can act as reservoirs of human influenza strains that circulated in the past, and also suggest that influenza A and B viruses may be transmitted from humans to seals as has been mentioned by other authors (Ohishi et al., 2002, 2004; Osterhaus, 2000). This transmission is due to the highly social lifestyle of pinnipeds, which congregate at sea and on land, and frequently associate with seals from other colonies. It is important to note that there is a strong interaction between seals and humans on Lobos Island and Polonio's Cape during live animal captures; nowadays, most interactions occur during capture and research activities, as well as in rehabilitation centers and sometimes with divers that swim near the seal islands. These events constitute opportunities for new influenza strains to jump between humans and seals, providing the potential for an epidemic event. Gaining information on the full spectrum of influenza viruses circulating in our seal colonies and detecting these viruses will remain an important task for its

As described previously, tuberculosis in pinnipeds had been recorded from the beginning of the twentieth century but it was not until mid 80's and later that several publications appeared with data from stranded animals and zoological collections. It is important to note that most records are from Otariids from the Southern Hemisphere, a single diagnosis is from a southern elephant seal and one from one native Northern Otariid species kept in

Despite the fact that the seal harvest in Uruguay extended for 200 years, there are no records of macroscopic lesions observed in animals, nor of tuberculosis diagnosis from the staff working in the capture, slaughter or processing of the by-products, that could contribute retrospective data to this disease in Uruguayan pinniped colonies. The disease is considered endemic because of the numerous cases diagnosed in wild animals and the high prevalence of South American seals kept in aquaria and zoos. Unfortunately, to date non epidemiological studies have been conducted in any of the seal colonies from the Southern Hemisphere which could indicate the prevalence of the disease in its natural environment.

Information presented in this work, including the isolation of *M. pinnipedii* in pups and juvenile seals, indicates the early transmission of the organism. However, it is difficult to establish the course of the disease or immune mechanisms that may develop in each individual to control the infection or, in other cases, allow individuals to act as healthy carriers for several years until some trigger factor determines the development of the

The most important mechanism of transmission of *M. tuberculosis*, and probably *M. pinnipedii*, is through the air by droplets produced when an individual with respiratory tract TB eliminates aerosolized microorganisms by coughing or sneezing. Large droplets fall

surveillance, outbreak control, and animal and public health.

captivity in the Northern Hemisphere.

In the early course of host-pathogen interaction, mycobacteria are phagocytized by alveolar macrophages. In case the macrophage cannot destroy the pathogen, it resides in a quiescent state with a relatively low multiplication until cell mediated immunity is compromised transiently or permanently. Among the risk factors for immune-compromise, are age, history of a spontaneous TB cure with residual fibrotic lesions, and the time elapsed since infection. Other medical conditions such as endocrine disorders, tumors, malnutrition and stress may influence the progression of infection to disease (Musser et al., 2000; Rider, 1999). This evolution of the disease is consistent with the findings by different authors who have isolated AFB in organs without lesions from pups and juveniles (Bruning-Fann et al., 2001), identified the presence of calcified granulomas in mediastinal lymph nodes in adult and some juvenile animals in captivity and documented tuberculosis lesions in older animals (Jurczynski et al., 2011, 2012).

Keepers and veterinarians at zoos, aquaria and rehabilitation centers are at increased risk of infection because of their extensive contact with the animals. The degree of risk depends on the type of accommodation and sanitizing procedures of the enclosures (Kiers et al., 2008; Thompson et al., 1993) in association with the presence of open tuberculosis cases. Given the zoonotic condition of the disease, it is important to take preventive measures in all personnel working near or with access to wild and captive pinnipeds (researchers, fishermen, seal hunters, rehabilitation centers, aquaria and zoos staff) (Kiers et al., 2008; Thompson et al., 1993) and to establish hygiene measures for reducing the chances of spreading the infection.

Since development of the disease is triggered under immunosuppressive conditions, it is important to assess which factors directly or indirectly affect the immune system and allow development of the disease in wild populations of the Southern Hemisphere. This also applies to other diseases that may be affecting the health status of wild colonies, particularly in *O. flavescens* from Uruguay, whose population is declining.

Further investigation is needed in order to establish the sources of zoonotic potential in the marine environment and to better understand the nature of health risks for sea mammals and human. Additional epidemiologic studies are also required, to investigate epizootic

episodes assessing its impact and to elucidate how these diseases spread among and within marine mammal's populations.

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 173

Bernardelli, A.; Nader, A. J.; Loureiro, J.; Michelis, H. & Debenedetti, R. (1990). Micobacteriosis en mamíferos y aves marinas. *Revue Scientifique et Technique de l' Office* 

Bernardelli, A.; Bastida, R.; Loureiro, J.; Michelis, H.; Romano, M. I.; Cataldi, A. & Costa, E. (1996). Tuberculosis in sea lions and fur seals from the south-western Atlantic coast. *Revue Scientifique et Technique de l' Office International des Epizooties*, Vol. 15, (September,

Blair W. R. (1913). Report of the veterinarian on the mammals: In Seventeenth Annual report of the New York Zoological Society. *New York Zoological Society*, (1913), pp. 73-74 Blanc, A.; Ruchansky, D.; Clara, M.; Achaval, F.; Le Bas, A. & Arbiza, J. (2009). Serologic Evidence of Influenza A and B Viruses in South American Fur Seals (*Arctocephalus* 

Boersma, P. D. (2008). Penguins as Marine Sentinels. *BioScience*. Vol 58, N° 7, (July, 2008), pp.

Bruning-Fann, C. S.; Schmitt, S. M.; Fitzgerald, S. D.; Fierke, J. S.; Friedrich, P. D.; Kaneene, J. B.; Clarke, K. A.; Butler, K. L.; Payeur, J. B.; Whipple, D. L.; Cooley, T. M.; Miller, J. M. & Muzo, D. P. (2001). Bovine tuberculosis in free-ranging carnivores from Michigan.

Callan, R. J.; Early, G.; Kida, H. & Hinshaw, V. S. (1995). The appearance of H3 influenza viruses in seals. *Journal of General Virology,* Vol. 76, (January, 1995), pp. 199–203 Campagna, C. & Le Boeuf, B. (1988). Reproductive behaviour of Southern Sea Lions.

Campagna, C. & Lewis, M. (1992). Growth and distribution of a southern elephant seal

Cappozzo, H. L.; Bernabeu, R. O. & Crespo, E. A. (1994). Discriminación de stocks de *Otaria flavescens* y *Arctocephalus australis* en Uruguay y Argentina. Sub Proyecto 1. Pp 1-9. In: *Problemas de conservación y manejo de los mamíferos marinos del Atlántico Sud Occidental en Uruguay y Argentina: un proyecto conjunto de investigación*. Programa de las Naciones Unidas para el Medio Ambiente (PNUMA). Informe final sobre los trabajos realizados

Castro-Ramos, M.; Ayala, M.; Errico, F. & Silvera, F. V. (1998). Aislamiento de *Mycobacterium bovis* en Pinnípedos *Otaria byronia* (Lobo marino común) en Uruguay. *Revista de* 

Castro-Ramos, M.; Leizagoyen, C.; Alves, F.; Cirillo, F. & Zipitría, R. (2001). Tuberculosis en *Arctocephalus australis* (Lobo marino de dos pelos) en la costa de Uruguay, América del Sur. *V Encuentro Nacional de Microbiólogos,* Noviembre de 2001. Montevideo, Uruguay. Castro-Ramos, M.; Katz, H.; Castro, O. & Gutiérrez, F. (2005). Primera comunicación de tuberculosis en *Mirounga leonina* (Mammalia, Phocoidea) (elefante marino del sur) en Uruguay. *VII Encuentro Nacional de Microbiólogos.* Octubre de 2005. Montevideo,

Castro-Ramos, M.; Katz, H.; Moraña, A.; Tiscornia, M. I.; Morgades, D. & Castro, O. (2006). Tuberculosis en pinnípedos (*Arctocephalus australis* y *Otaria flavescens*) de Uruguay. In: *Bases para la conservación y el manejo de la costa atlántica uruguaya*. Menafra R., Rodríguez-

*australis*). *Journal of Wildlife Diseases*, Vol. 45, N° 2, (April, 2009), pp. 519–521

*Journal of Wildlife Disease*s, Vol. 37, No. 1, (January, 2001), pp. 58–64

colony. *Marine Mammal Science*, Vol. 8, (October, 1992), pp. 387–396

*Behaviour*, Vol. 104, (January, 1998), pp. 233-261

*Medicina Veterinaria*. Vol. 79, No. 3, (1998), pp. 197-200.

*International des Epizooties,* Vol. 9, No. 4, (1990), pp. 1121-1129

1996), pp. 985-1005

durante 1992-1993

Uruguay.

597-607
